T-regulatory cells require Sin3a for stable expression of Foxp3

Histone deacetylases 1 and 2 play a major role in the transcriptional regulation of T-regulatory (Treg) cells via interactions with a myriad of coregulatory factors. Sin3a has been well established as a Hdac1/2 cofactor, while its role within Tregs has not been established. In this study, the effects of conditional deletion of Sin3a within Foxp3+ Tregs were evaluated. Developmental deletion of Sin3a from Foxp3+ Tregs resulted in the rapid onset of fatal autoimmunity. Treg numbers were greatly reduced, while residual Tregs had impaired suppressive function. Mice also showed effector T-cell activation, autoantibody production, and widespread tissue injury. Mechanistically, Sin3a deletion resulted in decreased transcription of Foxp3 with a complete lack of CNS2 CpG demethylation. In addition, Foxp3 protein stability was impaired with an increased ex-Treg population. Thus, Sin3a plays a critical role in the maintenance of Treg identity and function and is essential for the expression and stability of Foxp3.

In addition to TFs and cofactors, nucleotide and chromatin epigenetic modifications contribute greatly to the expression of Foxp3 and the regulation of the Treg transcriptome.Nucleotide modifications regulate gene transcription through DNA methylation; hypo-or hypermethylated CpG sites of promoter/ enhancer regions result in gene expression or silencing, respectively (12).CpG methylation takes place through DNA methyltransferase (Dnmt) activity, while CpG DNA demethylation occurs primarily through the actions of 10-11 translocation (Tet) methylcytosine dioxygenases (12).The extent of CpG methylation is of major importance to Foxp3 expression and the development of the Treg lineage (13)(14)(15).Enhancers conserved noncoding sequences (CNS1 and CNS2) of Foxp3 become completely demethylated upon Treg lineage commitment, supporting Foxp3 expression and stability, and Tet-mediated epigenetic regulation is essential to the maintenance of Foxp3 expression and Treg phenotype (16)(17)(18)(19).
Chromatin modifications regulate gene transcription via the alteration of nucleosome-forming histones.Histone modifications include methylation, phosphorylation, ubiquitylation, and various forms of acylation, including acetylation and crotonylation.Typically, histone acetylation occurs on lysine residues of histones and relaxes the chromatin (euchromatin formation), thus allowing TF and polymerase access to the DNA.Histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes are responsible for acetyl addition or removal, respectively.Various HATs, including Cbp, p300, Tip60, and Pcaf, contribute to transcriptional regulation within Tregs, including that of Foxp3 (20)(21)(22)(23)(24).Likewise, many HDACs are active within Tregs, with most having a direct effect on Foxp3, though with varying functional outcomes (25).Pharmacologic inhibition of HDAC activity can promote or impair Treg function, depending upon the HDAC target(s) (26,27).HDACs commonly function as members of large multiprotein complexes.HDAC1 and HDAC2 interact with and function within multiple coregulatory complexes, including CoREST, NuRD, and Sin3a.Mass-spec analysis has shown these coregulatory complexes are present within Tregs and are thereby expected to influence their production and/or function (25).However, the extent to which the Sin3a complex contributes to the regulation of Foxp3 and/or the Treg phenotype has been unknown.
In this study, we evaluated the role of the scaffolding protein, Sin3a, in Foxp3+ Tregs.We found that Sin3a regulates the transcription of a large and diverse set of genes, including those specific to Tregs.Importantly, Sin3a was found to support the expression and stability of Foxp3, along with the Treg phenotype and suppressive function.Hence, Sin3a plays a vital role within Tregs and could potentially be targeted in certain contexts, such as decreasing Treg function to thereby boost host immune responses in cancer (44).

Methods Mice and cell lines
We purchased Ai9(RCL-tdT) (stock No. 007909) and tamoxifen (TAM)-inducible Foxp3cre (stock No. 016961) mice from The Jackson Laboratory, in addition to previously described Foxp3 YFP- cre (45), CD4 cre (46), and Sin3a flox/flox (47) mice.Sin3a −/ − Foxp3 YFPcre mice and Foxp3 YFPcre controls were used 12-15 days after birth due to the early onset of lethal autoimmunity in mice with conditional deletion of Sin3a in their Tregs, whereas inducible knockout (KO) mice were used at 6-8 weeks.Mice were housed and handled in accordance with the IACUC (protocol No. IAC-22-000561) and The Children's Hospital of Philadelphia Research Institute policy.The 293T cell line used in this study was obtained from the ATCC repository (CRL-3216).

Histology
Sections of formalin-fixed, paraffin-embedded tissues were stained with hematoxylin and eosin (H&E) and analyzed in a blinded manner by a pathologist.

Detection of autoantibodies
Cryosections (4-5 mM) of unfixed normal tissues were incubated for 1 h at room temperature (RT) with Sin3a −/ − Foxp3 YFPcre sera, washed, incubated for 30 min at RT with FITC-labeled goat antimouse secondary Ab, washed, and mounted.Precharacterized sera from mice with known autoantibodies were used as positive controls, while pooled normal sera and secondary Abs alone were used as negative controls.

Cell dissociation and cryopreservation
Tissues collected from mice were homogenized using a pestle over 0.4 µm filter fabric, dissociated cells collected in cold PBS, erythrocytes lysed using ddH 2 O, and single-cell suspensions used in same-day experiments or cryopreserved (48).Throughout, mammalian cells were centrifuged at 300×g for 10 min at 4°C, unless otherwise specified.Cell numbers and viability were determined using a Nexcelom Cellometer Auto 2000 cytometer and slides with AOPI viability dye (Nexcelom Bioscience, Lawrence, MA, USA).For cell enumeration and flow cytometry analysis, eight subcutaneous lymph nodes and four mesenteric lymph nodes (LN), the spleen, and the thymus were collected and processed from each mouse.For experiments that utilized bead-isolated or sorted Tregs, the spleen and around 15 LN (secondary lymphoid organs [SLOs]) from each mouse were dissociated, and when necessary, as for Sin3a −/− Foxp3 cre mice, were pooled for processing.

Flow cytometry
Single-cell suspensions from subcutaneous and mesenteric LN, spleens, and thymi were prepared as described above.Single-cell suspensions of fresh or reconstituted cryopreserved cells were stained with fl uorophore-conjugated mAbs listed in Supplementary Table S1.Cells were stained with fixable viability dye for 15 min at RT, stained for cell-surface antigens for 45 min at 4°C, and, as necessary, fixed and permeabilized (BD, Pharmingen Transcription Factor Buffer Set, BD, Franklin Lakes, NJ, USA) according to the manufacturer's protocol prior to staining cytosolic and/or intranuclear targets for 1 h at 4°C.When staining for chemokine or cytokine production, cells were incubated with phorbol 12-myristate 13-acetate (PMA), ionomycin, and brefeldin for 3 h prior to fixation.For annexin V staining, cells were incubated in annexin-binding buffer (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol and stained with annexin V Ab (Supplementary Table S1).For fluorescence-activated cell sorting (FACS) staining, cells were centrifuged at 1,300 rpm for 5 min and kept in the dark (or limited light) as described in the current FACS staining guidelines (49).Data from stained cells were acquired on a Cytoflex (Beckman Coulter, Brea, CA, USA) flow cytometer and analyzed using FlowJo v10 software.

DNA methylation detection
Cells dissociated from SLOs of Sin3a −/− Foxp3 YFPcre and Foxp3 YFPcre controls were CD4+ and CD25+ enriched using magnetic bead isolation, then Foxp3 YFP +CD25 PE + Tregs were collected by FACS sorting.DNA was isolated from Foxp3+CD25 + Tregs using the Qiagen DNA Kit as per the manufacturer's protocol.Bisulfite conversion and desulfination of DNA (~300 ng/ sample) was done using the Methyl Detection Kit by Active Motif.The CNS2 promoter region (aka TSDR) of foxp3 was amplified using nested primer sets (outer set: "Foxp3_TSDR outer F" and "Foxp3_TSDR outer R" and inner set: "Foxp3_TSDR inner F" and "Foxp3_TSDR inner R") detailed in Supplementary Table S2 and described previously (52).The CNS2 PCR product was verified by and purified from agarose gel (1% agarose, 1× Trisacetate-EDTA (TAE), 1 k, and 100 bp ladder, stained with Ethidium Bromide (EtBr)), using the QIAquick Gel Extraction Kit as per the manufacturer's protocol.The converted CNS2 region was inserted into a TOPO2.1 TA vector using T4 DNA Ligase (Invitrogen) and transformed into TOP10 chemically competent Escherichia coli (Invitrogen) according to the manufacturer's protocol.Transformed E. coli were selected from LB agar plates containing ampicillin (100 mg/ml) and galactose (40 mm/ml).Plasmids were isolated (Invitrogen PureLink Quick Plasmid Miniprep Kit, following the manufacturer's protocol) from overnight cultures, and the presence of CNS2 inserts was confirmed via restriction enzyme digestion with EcoRI (NEB) followed by gel electrophoresis (1% agarose gel, 1× TAE, 1 k, and 100 bp ladder, stained with EtBr).The plasmids (meeting quality control measures of OD260/280 ≥ 1.8 and OD260/230 ≥ 2.0) were then sequenced (Sanger) using both forward (M13 Forward −21) and reverse (M13 Reverse −26) priming per sample (Supplementary Table S2) by the DNA Sequencing Facility at the University of Pennsylvania.Sequencing results were analyzed using methylKit packages in RStudio.Throughout these procedures, DNA concentration and quality were measured with a Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), and as necessary, Zymo Research DNA Clean and Concentrator Kit was utilized according to the manufacturer's protocol.

Coimmunoprecipitation
293T cells were cotransfected with Sin3a and either Foxp3, Hdac1, or Hdac2 plasmids using Lipofectamin 3000, according to manufacturer's protocol.After 48 h, cells were washed with cold PBS and lysed on ice with lysis buffer containing 20 mM Tris-Cl at pH 7.5, 1% Triton-X100, 150 mM NaCl supplemented with protease and phosphatase inhibitor cocktails in addition to 1 mM DTT, 1 mM EDTA, 1 mM PMSF (just before lysates preparation).Cells were sonicated for 5 min in an ice bath (50% efficiency), rotated at 4°C for 30 min, and centrifuged at 12,000 rpm for 12 min.Proteins were estimated from clear supernatants using BCA reagents, using BSA as a standard.Equal amounts of protein lysates were precleaned with 2 µg of nIgG/tube (mouse or rabbit, depending on the IP antibody) for 1 h.Prewashed 30 µl protein A/G magnetic beads were added to each tube and rotated for 30 min at 4°C . Using a magnetic separator, precleaned lysates were collected, and equal amounts of lysates containing ~300 µg total protein immunoprecipitated with 5 µg each of nIgG, Sin3a, Hdac1, Hdac2, or other antibodies for 2 h with rotation at 4°C.Next, 30 µl prewashed magnetic beads were added to each tube and rotated for 1 h at 4°C.The IP beads were separated in the magnetic separator and washed four times with 500 µl of lysis buffer each time.Finally, IP beads were heated at 100°C in 1× sample lysis buffer for 10 min.Immunoprecipitated proteins were separated by 4%-15% SDS-PAGE.Western blots were developed with primary antibody for 2 h, washed four to five times, and then light chain-specific second antibody-conjugated horseradish peroxidase (HRP) was used for development with ECL reagents.In some experiments, antirabbit secondary antibody-HRP conjugates were also used.

RT-qPCR and RNAseq
RNA was isolated from bead-isolated CD4+CD25+ cells of the CD4 cre background or from sorted CD25 PE +Foxp3 YFP + Tregs of the Foxp3 YFPcre background using TRIZOL reagent (Ambion, Waltham, MA, United States) according to the manufacturer's protocol.Sin3a −/ − Foxp3 YFPcre and Foxp3 YFPcre control Foxp3 YFP +CD25 PE + Tregs were sorted (FACS) following CD4+CD25+ enrichment by magnetic bead isolation (Miltenyi).For qPCR, mRNA was converted to cDNA using Reverse Transcriptase (Applied Biosystems, Carlsbad, CA, USA) with a BioRad C1000 thermocycler.TaqMan Gene Expression Master Mix (Applied Biosystems) was used with TaqMan primer sets listed in Supplementary Table S2 as per manufacturer recommendations.qPCR target amplification was detected using a StepOnePlus Real-Time PCR System by Applied Biosystems.Genes commonly used as experimental controls for transcription, such as HPRT and GAPDH, were differentially expressed in Sin3a −/− Tregs (Supplementary Figure S16B); therefore, 18S was utilized.Fold change was determined using the formula 2 −DDCt , where data were normalized to endogenous 18S and control target expression.Concentrations and quality of mRNA and cDNA were determined using a ThermoScientific Nanodrop 2000, and when necessary, RNA was cleaned and/or concentrated using the Zymo Research RNA Clean and Concentrator Kit according to the manufacturer's protocol.

Statistics
Statistical analysis and figure creation were done with Excel, RStudio, and/or GraphPad Prism software, unless otherwise stated.Experiments were performed with a minimum of three biological replicates in technical triplicates, unless otherwise specified.Parametric tests were applied if data were normally distributed, and nonparametric tests if not.Data are shown as the mean SEM.A two-tailed p-value of 0.05 or less was considered to be significant: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; and ****p ≤ 0.0001.

Conditional deletion of Sin3a in Foxp3+ Treg results in rapid development of lethal autoimmunity
To study the role of Sin3a in Treg cells, Sin3a fl/fl mice were crossed with Foxp3 YFPcre .Mice with homozygous deletion of Sin3a in Foxp3+ Tregs developed severe autoimmunity and died within 15-22 days of birth.These mice were smaller than normal littermates (Figure 1A), with ridged, dry skin and mucous membranes, and underdeveloped ears.Sin3a −/− Foxp3 YFPcre mice had markedly enlarged subcutaneous LNs (sLNs) and smaller spleens than littermate controls, while mesenteric LN (mLNs) were of normal size (Figure 1B); these differences were reflected in the number of cells recovered from dissociated organs (Figure 1C).Sin3a deletion within Tregs led to abundant tissue damage (Figure 1D; Supplementary Figure S1A).Histopathology of the lungs and livers showed loss of tissue structure, fibrosis, and edema, with immune cell infiltrates.Spleens displayed fibrosis and immune infiltrates, and skin showed tissue thickening, fibrosis, and immune infiltrates.Mice lacking Sin3a in Tregs produced abundant organ-specific autoantibodies including antikeratin Abs (AKA), antiparietal cells Abs (APCA), and antiendomysial Abs (EMA) (Figure 1E), and had heavy proteinuria (Supplementary Figure S1B).Together, these data showing extensive tissue injury and rapid death indicate that Sin3a perform a vital role in Treg cells, with consequences for prevention of autoimmunity.

Mice with conditional Sin3a deletion in Foxp3+ cells have fewer Tregs and more effector T cells
Flow cytometric evaluation of T-cell populations in lymphoid organs shows that, compared to WT controls, Sin3a −/− mice had more CD8+ T cells in their spleens (Figure 2A), more CD4+ T cells in their thymi (Figure 2B), and a marked reduction of Foxp3+ Tregs in SLOs (Figure 2C).Relations between control and Sin3a −/− T-cell numbers differed from FACS percentages in some cases, e.g., subcutaneous LN from Sin3a −/− mice were much larger (Figure 1B) and had more cells (Figure 1C) than controls.Within the massively enlarged sLN of Sin3a −/− mice, the actual number of Tregs did not differ from controls (Figure 2F), while the numbers of CD8+ (Figure 2D) and CD4+ (Figure 2E) T cells increased greatly.FoxP3+ Tregs were nearly depleted in the spleens and mLN of Sin3a −/− mice (Figure 2F), and CD4+ T cells were also reduced to near depletion in mLN (Figure 2E).In addition, within spleens and sLN, the ratios of CD4+ T cells to Tregs were much higher in Sin3a −/− mice (Figure 2G), illustrating a cellular imbalance toward an effector phenotype.T cells from the SLOs of Sin3a −/− mice were further examined using phenotypic markers for memory, naïve, and effector subpopulations (CD44 and CD62L).In the spleens and sLNs, but not mLNs, of Sin3a −/− mice, the CD4+ and CD8+ T-cell populations were composed of more effector (CD62Llo) and fewer naïve (CD44+CD62L++) cells as compared to controls (Supplementary Figures S2C-E).This phenotypic skewing of CD4

Conditional deletion of Sin3a in CD4+ T cells
The very low Treg numbers seen in Sin3a −/− Foxp3 cre mice markedly constrained ex vivo studies.We therefore deleted Sin3a in CD4+ cells, seeking to generate mice in which we could isolate Sin3a-deficient Tregs in sufficient numbers for further studies.The reasoning behind this was that Sin3a deletion from both suppressive and effector CD4+ T lineages would rescue the severe early-onset autoimmunity and fatality of Sin3a −/− FoxP3 cre deletion.Indeed, Sin3a −/− CD4 cre mice did not develop fatal autoimmunity.Rather, both CD4+ and CD8+ T-cell populations were drastically decreased, by percentage and cell numbers, in the SLOs of Sin3a −/ − CD4 cre mice (Supplementary Figures S4A, G).Within SLOs, proportions of Foxp3+ cells were markedly increased compared to CD4 cre controls (Supplementary Figure S4A).While incongruent with the percentages of FoxP3+ cells within mice with conditional Sin3a deletion from Foxp3+ Tregs, these results are consistent with a recently published analysis of Sin3a −/− CD4 cre mice (56).However, when considering cell numbers within these organs, Foxp3+ Tregs were actually reduced in the spleens and sLNs (Supplementary Figure S4G).Tregs and CD4+ cells had reduced naïve and elevated effector subpopulations in the spleens, subcutaneous and mesenteric LNs (Supplementary Figure S4D).Naïve CD8+ T cells were also reduced in the SLOs (Supplementary Figure S4D).Memory CD4+ and CD8+ T cells were drastically increased in the SLOs, while the Treg memory population increased only in the sLNs (Supplementary Figure S4D).Hence, as in the Foxp3 cre background, Sin3a −/− CD4 cre mice displayed a cellular imbalance toward an activated immune environment.

Inducible Sin3a deletion in existing Foxp3+ Treg cells
In further efforts to obtain larger numbers of ex vivo Foxp3+ Tregs lacking Sin3a, we utilized a tamoxifen-inducible Sin3a −/ − Foxp3 cre (Sin3a −/− TAM) model.This allowed mice to develop normally prior to Sin3a deletion from Foxp3+ Tregs.Following tamoxifen treatment (Supplementary Figure S5), mice did not succumb to fatal autoimmunity, but like Sin3a −/− Foxp3 cre mice, they had massive sLNs (Supplementary Figure S6A) with pronounced follicles and germinal centers and developed PMN infiltrates and airway cuffing in the lungs (Supplementary Figure S6B).T-cell populations were assessed by FACS and had no significant differences in percentages of CD8+, CD4+, or Foxp3+ T cells (Supplementary Figure S7A).However, there was a significant downward shift in Foxp3 fluorescent intensity in Sin3a −/− TAM Tregs (Supplementary Figure S7A), like those from CD4 cre (Supplementary Figure S4) and Foxp3 cre (Figure 2H) backgrounds.T-cell subpopulations were also similar to those of the other two genetic backgrounds studied, whereby CD4+ cells and Foxp3+ Tregs had increased effector and decreased naïve types, especially in sLNs (Supplementary Figure S7E).While there were differences in the percentages of Foxp3+ Tregs among Sin3a −/− mice of Foxp3 cre , TAM-induced Foxp3 cre , and CD4 cre backgrounds, in each case Foxp3 was reduced compared to controls.The CD25/Foxp3 FACS profiles illustrate the depletion of Foxp3 and the shift from CD25+Foxp3+ to CD25+Foxp3− in Sin3a −/− Foxp3 cre mice (Figure 2H).The great loss of Foxp3 protein within Sin3a −/− Tregs as determined by fluorescent intensity was substantiated by Western blot (Figure 2J).In addition, a massive reduction in transcriptional expression of Foxp3 was detected by RT-qPCR with both CD4+CD25+ bead-isolated Tregs of the CD4 cre background (Figure 2K) and CD25+FoxP3+ sorted Tregs of the Foxp3 cre background (Supplementary Figure S14B).Hence, deletion of Sin3a from Tregs resulted in the reduction of Foxp3 protein and altered immune cell populations, leading to varying degrees of immune homeostasis disruption.

Sin3a is important for Foxp3+ follicular Treg development
Given enlarged lymphoid follicles and germinal centers in sLNs Sin3a −/− mice (Supplementary Figure S6B), follicular immune cell populations were evaluated by flow cytometry.There were more follicular T helper (Tfh) cells in the SLOs of Sin3a −/− mice (Figure 3A), whereas follicular T-regulatory (Tfreg) cells were decreased in spleens and sLNs (Figure 3B).The ratios of Tfh to Tfreg cells were much higher in the spleens and sLNs of Sin3a −/ − mice (Figure 3C).Follicular B cells were unchanged (Figure 3D), while marginal zone (MZ) B cells (Figure 3E) and MZ precursor B cells (Figure 3F) were reduced in Sin3a −/− spleens and sLNs.In contrast, germinal center (GC) B cells were increased in Sin3a −/− spleens and sLNs (Figure 3G).While not exclusive to lymphoid follicles, B-regulatory cells were reduced in the sLNs of Sin3a −/− mice (Supplementary Figure S10).A few other differences were observed in B-cell subtypes (Supplementary Figure S9).These results show that Sin3a has a profound effect on the cellular composition of the germinal follicles of SLOs.

Sin3a is key to the transcriptional regulation of Foxp3+ Treg cells
We used RNAseq to better understand the extent to which Sin3a contributes to transcriptional regulation in Foxp3+ Tregs (Figure 4; Supplementary Figures S14-S16).There were hundreds of uniquely expressed genes in Tregs with and without Sin3a (Figure 4A).Among the genes differentially expressed between Sin3a −/− and control Tregs, thousands of genes had enhanced or  RNAseq analysis of sorted CD25+Foxp3+ Tregs from Sin3a −/− Foxp3 YFPcre or Foxp3 YFPcre control mice.Gene coexpression between groups is depicted in a Venn diagram (A).DEGs of Sin3a −/− vs. CTR Tregs with |log 2 fold change|≥ 1 and padj ≤ 0.05 in a volcano plot (B), and selected DEGs depicted by heatmap with log 2 fold change (FC) in the left column with the color scale to the right (C).GSEA output using DEGs of Sin3a −/− versus CTR Tregs (NES: − 1.338; p-value: 0.006; FDR: 0.156) (D).RNAseq 3D PC plot (Supplementary Figure S15A), fpmk distribution violin plot (Supplementary Figure S15B), and total DEG heatmap (Supplementary Figure S16A) are included in the Supplementary Materials.RNAseq was performed using six pooled biological replicates per group, three males and three females.
While transcriptional expression of Foxp3 and other Tregspecific factors was reduced in the absence of Sin3a, markers of other lineages and immune cell subtypes were enhanced, including T-bet, Blimp1, IRF4, CD63, CD8a, and CD8b1 (Figure 4C; Supplementary Figure S16).CD8a was the most significantly altered DEG (ranked by padj value), and this was confirmed by increased CD8a protein in Foxp3+ Tregs via FACS (Supplementary Figure S2F).Other CD8+ T-cell-associated factors such as CD160, EOMES, and Batf3 were also increased in transcriptional expression (Figure 4C; Supplementary Figure S16B).Gene Set Enrichment Analysis (GSEA) of DEGs revealed that CD4 and CD8 T cell differentiation (Figure 4D) and Th17 cell differentiation (Supplementary Figure S15M) were impacted by the absence of Sin3a.Myeloid lineage and inflammatory mediators were also increased in Tregs lacking Sin3a, including GM-CSF (Csf2), Batf2, NFAT5, CD14, CD33, and CD36.Conversely, the majority of DEGs of B-cell-associated factors such as CD5, CD37, CD38, and CD79a/b (B-cell receptor [BCR]) were reduced (Supplementary Figure S16B).Together, these results display a markedly dysregulated transcriptional profile of Foxp3+ Treg cells, specifically favoring proinflammatory effector mechanisms.
Enrichment analysis of DEGs identified immune processes and hematopoiesis to be significantly altered by the Sin3a deletion (Supplementary Figure S15D).Likewise, GSEA revealed many signaling pathways involved in immune processes, such as NODlike, NOTCH, MAPK, tumor necrosis factor (TNF), RIG I-like, and Toll-like signaling (Supplementary Figures 15E-K), were impacted.DEGs that were reduced or enhanced in Sin3a −/− Tregs were cross-referenced with ATAC-seq data from an open-access repository, and the top four results from each query are included in Table 1.DEGs with reduced expression in Sin3a −/− Tregs best-overlapped chromatin availability of Tregs.DEGs with enhanced expression in Sin3a −/− Tregs best-overlapped chromatin availability of dendritic cells (DC), followed by monocytes.These results show that Sin3a supports transcriptional activation of Treg-specific factors and repression of alternative hematopoietic cell types.Hence, the Sin3a scaffolding protein plays a pivotal role in the control of the Treg transcriptional profile.
Along with increased cytokine production in Tregs, CD4+ (Figure 5D) and CD8+ (Supplementary Figure S11C) T cells from the same tissues produced higher cytokine levels.CD4+ T cells from spleens and sLNs of Sin3a −/− mice produced more GrzB and IFN-g, and the spleens also produced more TNF-g (Figure 5D).CD8+ T cells from SLOs of Sin3a −/− mice produced more GrzB and IFN-g, while CD8+ T cells from spleens and sLNs produced more TNF-g (Supplementary Figure S11C).These results demonstrate an environment where activated and dividing peripheral T effectors are not being suppressed by hyperactivated and dysfunctional Sin3a −/− Tregs.

Sin3a helps maintain Foxp3 protein stability in Treg cells
Sin3a −/− Foxp3 cre mice had drastically depleted Foxp3+ Treg populations in their SLOs, while thymic populations remained similar or even higher than those in controls.To assess whether the low numbers of peripheral Foxp3+ Tregs were caused by the loss of Foxp3, resulting in the generation of exTregs, we utilized a R26_tdT lineage tracing model, illustrated in Supplementary Figure S17A.When Foxp3 YFP-cre R26tdT mice were compared to those with Sin3a deletion (Sin3a flox/flox Foxp3 YFP-cre R26tdT), mice with Sin3a deletion had higher percentages of R26+Foxp3− exTregs in the SLOs and thymi (Figure 6A).These data show that the The fate of peripheral Tregs lacking Sin3a.The percentage of R26tdT+Foxp3− (exTregs) within CD45+CD4+ cells in Sin3a −/− or control mice (A) with representative.fcsplots of the sLNs included to the right.Diagram of the genetics and flow-cytometry analysis of Treg fate mapping using the R26 STOP tdTomatoFoxp3 YFPcre model (Supplementary Figure S17A).The same samples were assessed for apoptotic cell death via flow cytometry staining for annexin V (AnxV) and Ghost-viability dye within CD45+CD4+R26+ cells (B) with representative FCS plots of sLNs to the right.The percent AnxV+Ghost− apoptotic CD4+CD25+ cells from Sin3a −/− CD4 cre or control lymphoid organs determined by FACS (C) with representative FCS plots from sLNs to the right.FCS plots which represent spleen, mLNs, and thymus data are included in Supplementary Figures S17B, C Frontiers in Immunology frontiersin.orgreduction of peripheral Foxp3+ Tregs in Sin3a −/− FoxP3 cre mice was at least due to the loss of Foxp3, resulting in an expanded exTreg population.cell death was also assessed as a potential contributor to the depletion of peripheral Foxp3+ Tregs in Sin3a −/− mice by FACS staining with annexin V (AnxV).Sin3a −/− Tregs from the sLNs displayed increased levels of AnxV binding in both Sin3a −/ − tdTR26Foxp3YFP cre (Figure 6B) and Sin3a −/− CD4 cre (Figure 6C) mice.RNAseq analysis of peripheral Sin3a −/− Tregs revealed many differentially expressed genes associated with the regulation of apoptosis.Proapoptotic factors such as caspases 3 and 12, Bak1, FasL, Apaf1, and cytochrome c were increased in transcript expression, while antiapoptotic factors like Bcl2, Noxa1, and Mcl1 were decreased (Supplementary Figure S14).There were, however, some pro-apoptotic factors with deceased expression, one of which was Bcl2l11 (aka Bim), as verified by RT-qPCR (Supplementary Figure S14A).This is of interest because Mcl1 and Bim have been described as the dominant anti-and proapoptotic factors (respectively) in Tregs (57,58).Overall, proapoptotic factors with increased expression outnumbered those with decreased expression, and vice versa.GESA-enriched DEG data illustrated a significant skew of expression variation in the p53 pathway (Supplementary Figure S15P) and ferroptosis (Supplementary Figure S15O).Thus, it is likely that one or more forms of programmed cell death contribute to peripheral depletion of Tregs, particularly in the sLNs of Sin3a −/− Foxp3 cre mice, in addition to exTreg formation.
In addition to exploring Foxp3 loss and apoptosis as reasons for the lack of peripheral Tregs in the absence of Sin3a, peripheral CD4 +CD25− cells were tested for the ability to induce Foxp3 production following IL-2, TGF-b, and CD3/28 stimulation.Deletion of Sin3a from the Foxp3 cre , TAM-induced Foxp3 cre , and CD4 cre backgrounds rendered CD4+CD25− cells practically incapable of Foxp3 induction (Figure 6D; Supplementary Figure S18A).Furthermore, selamectin treatment of CTR CD4+CD25− cells reduced Foxp3 induction in a dose-dependent manner (Supplementary Figure S18B).Together, these data suggest that peripheral reduction of Foxp3+ Tregs in Sin3a −/− mice is due to the combinatorial effects of programmed cell death, Foxp3 instability, and impaired Foxp3 induction.

Sin3a supports Foxp3 expression within Treg cells
Among the four Foxp3 regulatory regions, CNS2 (aka TSDR) has been specifically associated with the stability of Foxp3 expression (59).We questioned whether CNS2 accessibility due to CpG methylation was altered in Tregs lacking Sin3a, thereby destabilizing Foxp3 expression.Bisulfite conversion and sequencing of the CNS2 region of Foxp3+ Tregs revealed that in the absence of Sin3a, CNS2 remained nearly completely methylated while all CpG sites of the control CNS2 were demethylated (Figure 7A).The stark difference in CpG methylation of CNS2 between Sin3a −/− and control Foxp3+ Tregs suggests a massive alteration in methyltransferase activity.Tet enzymes (Tet1, Tet2, and Tet3) are DNA methyltransferases that contribute to the demethylation of Foxp3 regulatory regions (15,60).RNAseq analysis showed that Tet1 and Tet3, but not Tet2, had reduced expression in Sin3a −/− Tregs (Figure 7C), which was confirmed by RT-qPCR (Supplementary Figure 14B).The expression of Dmnt1, a methyltransferase known to methylate CNS2 (15), was increased in Sin3a −/− Tregs (Figure 7C).
ChIP-seq analysis (ChIP-Atlas) showed colocalization of FoxP3 with Sin3a in Tregs with H-H peak intensities with a STRING binding score of 500 (61), suggesting these molecules may interact as regulatory cofactors.Upon the co-IP pulldown of Sin3a and Foxp3, Sin3a did indeed interact with Foxp3 (Figure 7B), though there remains a possibility that other molecules contribute to this interaction, as benzonase treatment was not used during protein preparation.These data suggest Foxp3 and Sin3a may contribute to the regulation of the Treg transcriptional profile via direct or indirect interaction (likely accompanied by other cofactors), consistent with our overall data showing that Sin3a is required for the transcriptional regulation and maintenance of Foxp3 expression and Treg suppressive function.

Discussion
Foxp3+ Treg cells have an essential role in immune homeostasis as a result of two main functions: restriction of T effector cell activity and maintenance of self-tolerance.Both functions were impaired by Sin3a depletion in Foxp3+ Tregs.The pathology associated with severe and fatal autoimmunity observed in mice with conditional deletion of Sin3a in Foxp3+ Tregs closely resembled that of Scurfy mice, which have dysfunctional Tregs due to a frameshift mutation of Foxp3 (5,7).Treg suppression of Tcell proliferation in vitro was largely abrogated by the Sin3a deletion (Figure 5A; Supplementary Figure S13).In addition, mice with Sin3a deletion in Foxp3+ Tregs had fewer naïve and more activated (CD69+) effector T lymphocytes (Figure 5C; Supplementary Figure S3E), suggesting a lack of suppressive constraint by Tregs.Failure of self-tolerance was evident in Sin3a −/− Foxp3 cre mice by systemic tissue damage (Figure 1; Supplementary Figure S1) and abundant autoantibody generation (Figure 1E).Thus, Sin3a is crucial for Treg immunosuppressive functions.
When Sin3a deletion in Foxp3+ Tregs was induced following normal pre-and postnatal development (Supplementary Figure S5), evidence of autoimmunity developed in sLNs and lungs (Supplementary Figures S6A, B).Though the percentage of Foxp3+ Tregs was unchanged (Supplementary Figure S7A), Foxp3 protein levels decreased (Supplementary Figure S7A), as did Treg suppressive function (Figure 5A), and sLN of these mice had less naïve and more activated (CD69+) effector T cells (Supplementary Figures S7C, E).One explanation for why the induction of Sin3a deletion did not provoke the severity of autoimmunity present in Sin3a −/− Foxp3 cre is that the role of Sin3a within Foxp3+ Tregs is of vital importance during fetal and postnatal development.It is also likely the case that TAM-induced Sin3a deletion does not achieve full deletion in all Foxp3+ Tregs.Factors that could contribute to this include the variability in tamoxifen uptake and processing among cell and tissue types.Continued thymic production of Foxp3+ Tregs subsequent to tamoxifen treatment may also contribute to mixture of dysfunctional Sin3a −/− Tregs and functional Sin3a+ Tregs.Consistent with this, female heterozygous Sin3a −/+ Foxp3 mice did not develop fatal autoimmunity.
While the vast majority of our data supports that Foxp3 is reduced in the absence of Sin3a, the percentage of Foxp3+ Tregs within the CD4+ population was greatly increased in Sin3a −/ − CD4 cre SLOs (Supplementary Figure S4A), as also noted in the recent study by Perucho et al. (56).However, the interpretation of data regarding FoxP3+ Tregs from a CD4 cre KO background should consider the impact of KO within the other T lymphocyte lineages.Sin3a deletion from the CD4 cre background results in Sin3a deletion from that and all subsequent T lymphocyte lineages, including CD8 T, NK T, Th17, and Treg cells.Sin3a is present within such a T-cell lineage, and thus its absence would likely impact each of these cell types.Hence, it is difficult to draw conclusions regarding Tregs with respect to other CD4-lineage T lymphocyte populations in CD4 cre KO mice.Rather, Sin3a −/− Foxp3 cre mice were utilized for the analysis of cellular populations, where the percentage of Foxp3+ cells (Figures 2C, 3B) and the number of Foxp3+ Tregs (Figure 2F) were depleted.Our data are supported by the reduction of Foxp3 protein (Figures 2H, J) and transcript expression (Figures 2K, 4C; Supplementary Figures S14, S15B) and the increase of exTregs (Figure 6A) present in Sin3a −/− mice.Together, these provide a consensus that Foxp3 is indeed diminished in Sin3a-deficent Tregs.
Phenotypic changes in the CD4-lineage populations of Sin3a −/ − CD4 cre mice likely explain why they did not develop fatal autoimmunity like Sin3a −/− Foxp3 cre mice despite having dysfunctional Tregs.Evidence of this within the CD4 and CD8 Tcell populations is included in the Supplementary Materials, where the CD4 and CD8 T-cell populations shifted to increased effector and memory phenotypes and reduced naïve T cells (Supplementary Figure S4D).The CD4 T cells from Sin3a −/− CD4 cre mice had higher percentages of Ki-67+ division and CD69+ activation (Supplementary Figure S4C) compared to controls.Overall, the CD8 and the CD4 T cells in Sin3a −/− CD4 cre mice mainly showed an activated phenotype, similar to Sin3a −/− Foxp3 cre CD4 T-cell populations and consistent with reduced suppression by dysfunctional Tregs.However, unlike in Sin3a −/− FoxP3 cre , the percentages (Supplementary Figure S4A) and numbers (Supplementary Figure S4G) of CD4+ and CD8+ T cells in the SLOs of Sin3a −/− CD4 cre mice were greatly reduced, providing an explanation as to why these mice do not develop fatal autoimmunity.
An inconsistency remains, however, that Sin3a −/− CD4 cre mice maintained the ability to induce Foxp3 expression peripherally (Supplementary Figure S4A) while Sin3a −/− Foxp3 cre (Figures 2C,  F) did not.Induction of Foxp3 expression is facilitated through the CNS1 enhancer, which is dispensable for Foxp3 expression stability (62).Demethylation of CNS1 can occur via Dnmt3 (Dnmt3a and/or Dnmt3b) activity (13, 63), which has not been linked to Sin3a and is not differentially expressed in Tregs lacking Sin3a (Supplementary Figure S16A).Thus, in the absence of Sin3a, CNS2 remains methylated (Figure 7A), and Dnmt3 demethylation of CNS1 allows induction but not stability of Foxp3.This would presumably occur in Tregs of both CD4 cre and Foxp3 cre backgrounds, the difference in Sin3a −/− CD4 cre mice being the reduction and phenotypic variation of T-cell populations.This  corresponds with the concept that Sin3a is important to maintenance of Foxp3 expression through CNS2 demethylation.Hypomethylation of the Foxp3 enhancers CNS1 and CNS2 is required for Foxp3 expression initiation and stability.The role of methyltransferase Dnmt1 in CpG methylation of the Foxp3 enhancers is well established (13,52).Previous reports in other cell types have found that Sin3a regulates CpG methylation indirectly by DNMT1 repression and TET1 activation (38,64).In addition, "genome-wide" Dnmt1-mediated CpG methylation occurred in the absence of Sin3a (38).In Tregs lacking Sin3a, expression of Dnmt1 was dramatically increased, along with increased expression of two Dnmt1 (Figure 7C) cofactors, PCNA and UHRF1 (Supplementary Figure S16B).While the increase of Dnmt1and its cofactors likely contribute to the hypermethylation of CNS2, its continued state of hypermethylation suggests another layer of dysregulation.During T lymphocyte hematopoiesis the CNS2 region is methylated in CD4 T cells and must undergo CpG demethylation upon Treg lineage commitment (65).Therefore, a failure in de novo CpG demethylation may also be involved in Tregs lacking Sin3a.
RNAseq analysis of Foxp3+ Tregs (Sin3a −/− vs. Foxp3 cre CTR) revealed vast differential expression of genes spanning diverse functional associations.Significantly, the Sin3a deletion affected genes specifically associated with the Treg phenotype.In the absence of Sin3a, expression of markers important to Tregs, including CD4, GITR, Helios, and Foxp3, was reduced, as were transcription factors involved in Foxp3 activation and phenotype (Runx1, Est-1, Rel, Jun, Fos, Bach2, and Foxo1), whereas factors associated with eTregs, such as Blimp1, IRF4, GrzB, Prf1, and IL-10, had increased expression.In addition, there were many genes typically expressed by other immune cells, lymphoid and myeloid, that were increased in Sin3a-deficient Tregs, including T-bet, CD8a, and IFN-g (Figure 8B).Thus, Sin3a is crucial to the regulation of the transcriptional profile of Foxp3+ Tregs.This conclusion is strengthened by the cooccurrence of DEGs downregulated in Sin3a −/− Tregs and the ATAC-accessible genes of Tregs (Table 1).Sin3a likely acts synergistically with Foxp3 to achieve Treg transcriptional identity.Most importantly, Sin3a supports Treg identity and function through Foxp3 expression stability via CNS2 demethylation.
The coregulator, Sin3a, plays a pivotal role in the regulation of transcriptional expression within Foxp3+ Tregs.This regulation spans a vast and diverse variety of functional networks, including those intimately linked to T-regulatory cell phenotypes.A central component of this was the extreme reduction of Foxp3 in Tregs that lacked Sin3a and the reduction of peripheral Foxp3+Tregs in Sin3s −/− Foxp3 cre mice.This peripheral depletion of Foxp3+ Tregs was likely caused by the loss of Foxp3 expression and ex-Treg formation due to CNS2 CpG-methylation, along with cell death, as seen particularly in sLNs.Sin3a deletion from Foxp3+ Tregs rendered the cells hyperactivated, proinflammatory, and lacking suppressive function.Hence, Sin3a is crucial for the stability of Foxp3, the control of effector activation, and the maintenance of Treg function.

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FIGURE 7CpG site methylation of the CNS2 promoter region of the foxp3 gene in CD25+Foxp3+ Tregs (A), where empty circles represent nonmethylated CpG and filled circles represent methylated CpG,and each row depicts an individual sample.Individual samples represent pooled DNA isolated from sorted CD25+Foxp3+ Tregs from SLOs of Sin3a −/− or Foxp3 YFPcre mice.Co-IP of Sin3a and FoxP3 in transfected 293T cells (B).DEGs determined by RNAseq of Sin3a −/− versus Foxp3 cre control CD25+Foxp3+ Tregs that are associated with Tet1 within the STRING network are displayed as circular nodes colored on a scale according to log 2 fold change (FC) and organized using Cytoscape MSC clustering (C).RNAseq was performed using six biological replicates per group, three males and three females.

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FIGURE 8 Proposed mechanism of Sin3a in T-regulatory cells.In a normal T-regulatory (Treg) cell, Sin3a facilitates CpG demethylation of CNS2 (TSDR) of the Foxp3 gene via TET1 and/or TET3, thereby supporting the stability of FoxP3 expression and lineage-specific factors (A).In T-regulatory cells lacking Sin3a CpG demethylation of the Foxp3 enhancer region, CNS2 does not occur, resulting in Foxp3 depletion and Treg dysregulation (B).Tregassociated transcription factors are depicted as yellow ovals and backgrounds, while those associated with alternate lymphoid lineages are represented as grey ovals and backgrounds.Genes with enhanced expression in the absence of Sin3a are represented by red text, while those with reduced expression are represented by blue text.

TABLE 1
Cooccurrence enrichment analysis of Sin3a −/− Treg DEGs and chromatin availability in immune cells.